Maintaining the overall title of the grant, the focus of the work will shift to later phases of B cell development, reaching from the naive, mature B cell to the germinal center (GC) reaction and memory B cell generation, maintenance and response. In addition, while we expect to bring the analysis of the role of BCR specificity in the formation and maintenance of the Bl and B2 subsets to completion within the first funding period, we expect that the experiments proposed for the extension period will further contribute to the understanding of this problem by identifying critical intracellular signaling cascades. Our work in the first funding period has demonstrated that the maintenance of mature B cells requires, apart from an NFicB-mediated signal through the BAFF-R (Sasaki et al., 2006), a maintenance signal through the BCR, involving the Igo/p cytoplasmic tail (Kraus et al., 2004). However, the molecular nature of this signal has remained undefined. Aim 1 of our proposal for the extension period attempts to clarify this issue, using targeted mutagenesis of the ITAM motifs in the cytoplasmic tails of the Igct/p heterodimer in combination, conditional inactivation of the phosphatase SHP-1, and a combined genetic conditional gain- and loss-of-function approach, in which the BCR isconditionally ablated in mature B cells, together with the activation of candidate intracellular signaling pathways like PI3 and MAP kinase signaling. We have generated vectors encoding constitutively active components of these pathways and are in the process of targeting them into the ROSA26 locus, according to a strategy, which we have developed for the induction of constitutive NFicB signaling (Sasaki et al., 2006). This part of the project connects our work to the areas of B cell subset differentiation as well as the pathogenesis of B cell lymphomas, where the activation of certain signalingpathways plays a critical role. Aim 2 addressesthe role of BCR signaling in T-independent and -dependentB cell activation and in particular the GC reaction. Our work before and in the present funding period (Kraus et al., 2001;Patterson et al., 2006) has established that conserved ITAM and non-ITAM tyrosines in the Iga cytoplasmic tail play distinct roles in T-dependent and -independent B cell activation. While our analysis of the non-ITAM tyrosines has come to a conclusion, the ITAM tyrosines appear to play an as yet enigmatic dual role, in that in combination with the Ig(3 ITAM they appear be crucial for BCR signaling altogether (Gazumyan et al., 2006;Kraus et al., 2001), whereas their mutation by themselves results in B cell hyper-reactivity upon BCR cross-linking. We hypothesize that the Iga ITAM tyrosines are essential for BCR signal-initiation as well as -termination, and that the former function is redundant with the ITAM tyrosines ofIgp\ Accordingly, our experiments aim at the identification of a negative feedback loop targeting the Iga ITAM tyrosines in BCR signal termination, using a biochemical approach with subsequent genetic verification. We will also assess a possible role of the Iga ITAM in antigen presentation, given the reduced T-dependent response of B cells harboring a mutated Iga ITAM (Kraus et al., 2001). Using GC B cell-specific gene targeting (Casola et al., 2006) and the various conditional alleles we have generated, we will assess the role of the Iga/p* heterodimer and its ITAMs and of BAFF-R in the control of the GC reaction in vivo. Aim 3 addressesthe role of BCR signaling in the maintenance and responsiveness to antigenic challenge of memory B cells, an issue that has remained largely unexplored in the past. We will address this issue using a cell transfer system, which we have recently established and which allows the selective tracing of genetically labeled memory B cells in vivo, together with conditional alleles of components of the BCR and systems of inducible gene targeting, which we have established in the past. On this basis, we will assess the role BCR expression and signaling as well as of BAFF-R in memory B cell maintenance. A particular focus will be on the relative contributions of the Iga/p heterodimer on the one hand and the cytoplasmic tails of IgH chains of y subclasses on the other, given that the BCRs of most memory B cells carry IgH chains with extended cytoplasmic tails, in contrast to the BCRs expressed by naive B cells. We have already constructed mouse mutants expressing Igyl chains or Ign chains with a y2b cytoplasmic tail instead of Igm chains, and are in the process of generating a conditionalallele allowing Cre recombinase-mediated replacement of an lygl chain by a genetically labeled tailless mutant. Finally, if memory B cell maintenance will turn out to be BCR dependent, we will use the combined gain- and loss-of-function approach described under Aims 1 and 2 to identify signalingpathways downstream of the BCR that contribute to cellular maintenance.